The development of effective and reliable multimolecular devices such as receptor-activated drug delivery systems molecular-scale sensors, switches, transducers and actuators requires control over the relative position of molecules within multimolecular structures. Molecules may be connected within multimolecular structures by covalent attachment (i.e., chemical bonds) or noncovalent means, including self-assembly, specific binding, hybridization of complementary nucleic acid sequences, ionic bonding, hydrophobic interactions, intercalation, chelation and coordination of metals. However, precise, reproducible and scalable methods for production of useful synthetic multimolecular devices with positional control at the molecular scale have heretofore been lacking.
Biological systems perform intricate functions through sophisticated molecular organization of complex molecules such as enzymes, antibodies, transmitters, receptors and regulatory proteins. Such intricate functions include signal transduction, information processing, cellular replication, growth and differentiation, biosynthesis, detoxification and transduction of chemical energy into heat and work. Wound healing, blood coagulation, muscle contraction, hormone secretion and complement-mediated immunity, for example, all represent biological functions that depend on multi-tiered cascades of biochemical reactions performed by organized molecules. Transport of ions and metabolites, gene expression and protein assembly represent a few of the many cellular functions that rely on concerted interaction of multiple organized biochemical systems. Efforts to simulate the productivity and efficiency of biomolecular machinery have been only marginally successful because of the inability to recreate the structural organization of molecules and groups of molecules inherent in highly ordered biological systems.
Biological systems have evolved two major capabilities that enable molecular manufacturing and nanomachinery far more sophisticated than chemical and biochemical methods developed by man. First, they have mastered the art of self-assembly, wherein discrete molecules either spontaneously organize or are chaperoned into supramolecular assemblies that perform complex functions through concerted interaction of the constituent molecules. Second, the rate and direction of biological reactions is manipulated through compartmentalization of reactants, catalysts and products, most commonly through physical segregation by cellular or subcellular membranes.
Efforts to develop self-assembling systems and microcompartmentalized biochemical reactions have escalated over the past several years. Historically, experimental approaches to self-assembly have been modeled after spontaneous association of lipids into monolayers and bilayer membranes. More recently, self-assembly has been attempted using lipid-protein mixtures, engineered proteins, branched DNA, and supramolecular chemistry.
Compartmentalization has been attempted through a wide range of approaches, including liposomes, microimmobilization techniques (e.g., photolithography) and targeted delivery (e.g., therapeutic immunoconjugates). Microscopic arrays of peptides and oligonucleotides have been achieved through light-directed combinatorial in situ synthesis on silicon substrates. However, the resolution of this technique is about a million-fold inadequate for ordered molecular arrays. Discrete resolution and manipulation of matter at the atomic level is being pursued through scanning tunneling microscopy and atomic force microscopy, but these techniques have not been developed for production-scale preparation of molecular arrays.
In a related area of bimolecular engineering, several types of bifunctional or hybrid molecules have been developed for diagnostic imaging and targeted drug delivery. Some of these include: chimeric antibodies, particularly humanized antibodies designed to eliminate human anti-mouse immune responses upon in vivo administration; bispecific antibodies, produced through enzymatic digestion of parent antibodies and controlled reconstitution using Fab fragments obtained from two different parents; conventional immunoconjugates, composed of a drug, toxin or imaging agent covalently attached or chelated to an antibody or antibody fragment through established immunochemical methods; and fusion proteins, most commonly immunotoxins for cancer therapy, generated from hybrid genes developed and expressed through recombinant methods. While these hybrid molecules, especially fusion proteins, provide a practical approach to controlled production of hybrid gene products, none of the above methods provides a unified approach to directed multimolecular assembly.
Many methods have been described for site-directed attachment of effectors (e.g., enzymes, isotopes, drugs, fluorophores) to antibodies, antigens, haptens and nucleic acid probes. However, these methods represent bulk techniques that do not provide sufficient specificity for reproducible preparation of ordered molecular pairs, groups or arrays. Further, while these methods enable production of bifunctional conjugates, they do not provide for concerted interaction between the constituent moieties (e.g., probe and reporter molecules).
Branched DNA has been used as a carrier for accommodating large numbers of enzyme labels (e.g., alkaline phosphatase), thus enabling biochemical amplification of specific binding reactions in diagnostic assays. Scientists investigating branched DNA as a three-dimensional structural design system have speculated that natural mechanisms by which drugs and particular proteins recognize and bind to specific sites on DNA could be applied to attach molecular electronic components to DNA for development of memory devices (Seeman (1993) Clin. Chem. 39:722). Seeman has also suggested attaching conducting polymers, such as trans-polyacetylene or polyphenothiazine, a PTL-ruthenium switch, and a redox bit into branched DNA structures. However, they have not suggested using a single oligonucleotide or hybridized pairs of oligonucleotides for coordinated placement of two or more different molecules within a single DNA structure. They also have not suggested selecting or engineering nucleotides to achieve requisite affinity for molecules that have no natural mechanism for recognizing specific sites on DNA.
Recognition and self-assembly are the two critical properties of chemical structures being explored in the rapidly advancing field of supramolecular chemistry. This field focuses on the designed chemistry of intermolecular bonds. For example, 12-crown-4-ether contains a central cavity that is highly specific for lithium. In fact, the components of this ring structure will self-assemble when exposed to a solution of lithium. Crown ethers and related structures are being investigated for their utility as highly selective sensors, sieves, synthetic enzymes and energy transfer structures for use in artificial photosynthesis. Other emerging applications include molecular switches, diodes, transistors and molecular wires, and it has been proposed that supermolecule interactions on thin films may enable computers to be built around liquid-phase assembly reactions.
A general method is described in Cubicciotti, U.S. Pat. No. 5,656,739 which provides for controlled placement of two or more selected molecules in appropriate spatial proximity to produce cooperative molecular assemblies. This method yields self-assembling multimolecular heteropolymeric complexes through use of synthetic heteropolymers or multivalent heteropolymeric hybrid structures comprising nucleotides having defined sequence segments with affinities for identified molecules.
Cubicciotti, U.S. Pat. No. 5,656,739 describes the advantages of synthetic oligonucleotides as assembly templates. Template-ordered molecules cooperate when brought into close spatial proximity, much like ordered biological molecules in living systems. Nucleic acids are particularly useful assembly templates not just because they can be selected to specifically bind nonoligonucleotide target molecules with high affinity (e.g., Tuerk and Gold (1990) Science 249:505-510), nor because they can hybridize by complementary base pairing. More important, only nucleic acids are capable roth of hybridizing other nucleic acids and specifically binding nonoligonucleotide molecules. Both forms of recognition can be programmably synthesized into in a single molecule or hybridized into a single discrete structure. A single nucleic acid molecule with two different binding specificities (i.e., a synthetic heteropolymer) can be synthesized at the push of a button and two or more synthetic heteropolymers can be hybridizably linked to one another.
Nucleotide-directed molecular assembly provides a general solution to the problem of molecular positioning by exploiting several key attributes of synthetic oligonucleotides. First, oligonucleotides can be designed or selected, e.g., by combinatorial methods, to specifically bind molecules of nearly any size and shape with high affinity, not simply other nucleic acids as once thought. Second, the informational properties of nucleotides enable reproducible synthesis of single oligonucleotides having two or more specific binding sites in defined spatial proximity within a single molecule. Third, the base pairing properties of nucleotides enable the splicing of any two useful binding sequences into a single discrete structure (i.e., a bifunctional hybrid structure) by programmable self-assembly (i.e., hybridization). Fourth, oligonucleotides comprising modified nucleotides can be used to attach selected molecules (e.g., ligands, receptors, structural or effector molecules) at the 3' or 5' ends or at defined positions along the nucleotide sequence. Multivalent assembly structures can therefore be designed to specifically recognize different effector molecules and position them to perform cooperative functions such as energy transfer, signal transduction, multistep enzymatic processing, molecular sensing, molecular switching and targeted or triggered molecular delivery, release and/or activation, e.g., as particularly useful in drug delivery. Designer oligonucleotides can be cost-effectively produced at large scale using automated synthesizers, and they can be conveniently attached to surfaces and nanostructures to permit self-assembly of immobilized devices and on-chip molecular arrays.
Unnatural bases and modified nucleotides comprising synthetic oligonucleotides are useful as diagnostic reagents, molecular biology tools and probes of nucleic acid structure and function (e.g., Goodchild (1990) Bioconjugate Chemistry 1:165-187;Beaucage et al. (1993) Tetrahedron 49:1925-1963). Prior art modified nucleotides include natural bases linked by spacers arms to molecular reporters (e.g., spin labels, fluorophores, quenchers, DNP, digoxigenin and biotin) and analogs designed to enhance duplex stability and chemical stability. Novel bases (i.e., analogs) include unnatural nucleotides designed to increase coding diversity (e.g., Piccirilli et al. (1990) Nature 343:33-37. Nucleic acids are useful materials for programmable self-assembly, because the bases and backbone can be extensively modified without compromising molecular recognition properties, stability or hybridization rates and without destroying the relatively rigid structure of short duplex oligonucleotides. Several nucleotide positions can be modified by addition of tethered substituents without significantly affecting duplex structure (e.g., the N2 and N7 positions of guanine, the N6 and N7 positions of adenine, C5 position of cytosine, thymidine and uracil, and the N4 position of cytosine).
It is well known that nucleotides can be modified by covalent attachment of ligands (e.g., DNP, digoxigenin, biotin) and receptors (e.g., antibodies), but the art is silent with respect to use of nucleotides as positioning devices for attachment of multiple specific binding pairs in suitable juxtaposition to enable functional coupling between, e.g., two specifically bound effector molecules.
The instant invention describes nucleotide-based and nonnucleotide multimolecular structures and multimolecular devices capable of positioning at least two specific recognition pairs (e.g., a pair of specific binding pairs, optionally including at least one shape recognition pair) within close spatial proximity (i.e., within functional coupling distance). extends the teachings of Disclosed herein are molecular templates comprising, imprinted from and/or mimicking multivalent nucleotides capable of positioning and functionally coupling multiple nucleotide or nonnucleotide molecules, at least one being a selected nonoligonucleotide molecule, to provide nucleotide-based and nonnucleotide multimolecular switches, multimolecular transducers, multimolecular sensors, molecular delivery systems, drug delivery systems, tethered recognition devices, molecular adsorbents, molecular adhesives and molecular adherents. Commercial applications include, e.g., therapeutics, diagnostics, cosmetics, agriceuticals, nutraceuticals, industrial materials, consumer electronics, molecular-scale batteries, packaging, environmental remediation, sensors, transducers and actuators for aeronautic and military use, smart polymers, adsorbents, adhesives, adherents, lubricants, biomimetically functionalized organic and inorganic semiconductors and carbon-based, silicon-based and gallium arsenide-based membranes, devices and systems. Nucleotide-based templates can be designed to recognize structural molecules comprising, e.g., surfaces, parts, products and packaging materials for use as willfully reversible and reusable molecular adhesives, adherents and adsorbents) and even biological surfaces. For example, template-directed delivery of selected molecules to keratin comprising hair and nails enables precise and specific, willfully reversible, application of safe, lasting, yet reversible cosmetic dyes, pigments, liners and structural elements. Selection of ligands, receptors, aptamers and shape recognition partners from diverse sequence, chemical and shape recognition libraries enables novel cosmeceutical formulations capable of specifically decorating, strengthening, protecting, lengthening and thickening hair, nails, eyebrows and eyelashes.
Templates comprising, e.g., synthetic heteropolymers and multimolecular devices may also be used as dopants, additives, active ingredients or smart polymers comprising commercial chemicals, materials, products and packages, particularly polymers, gels, foams, woven and nonwoven fibers, plastics, papers, rubbers, coatings, coverings, paints, powders, sealants, adhesives and even recycled materials, particularly as smart polymers capable of performing useful functions. Useful functions include, for example, stimulus-responsive molecular delivery, switching, sensing, transducing, and actuating changes in the internal or external environment or, alternatively, in the properties of the host material (e.g., shape, color, temperature, conductivity, porosity, rigidity, adhesiveness, odor).
The ability to intimately combine within a single multimolecular structure at least two specific recognition pairs with different specificities (i.e., with control over the relative positions of or distance between constituent molecules) enables the design and construction of molecular-scale devices including multimolecular switches, sensors, transducers, molecular delivery systems, adsorbents, adherents, adhesives and lubricants. Multivalent molecular structures of the instant invention enable controlled positioning and optionally covalent crosslinking of multiple specific recognition pairs within suitable intermolecular proximity to provide functional coupling between members of the recognition pairs. Selected effector molecules can be conjugated to defined positions of nucleotide or nonnucleotide scaffolds to enable both controlled intermolecular positioning and functional coupling of conjugated effector molecules and recognition pairs. Selected molecules positioned by specific recognition using affinity-based templates can subsequently be permanently or pseudoirreversibly attached to one another or to the template using well known chemical and enzymatic methods, e.g., covalent crosslinking reagents, ligases and synthetases. Alternatively, template-ordered molecules can be used as imprintable hosts for cast-and-mold printing of nonnucleotide (e.g., plastic) templates and assemblies shaped by templated guest molecules. Two members of a specific binding or shape recognition pair or even two different specific recognition pairs can be tethered by pseudoirreversible (e.g., covalent, avidin/biotin-based, or hybridization-based) incorporation within a nucleotide-based, aptameric, heteropolymeric or nonnucleotide device in such manner that specific binding and unbinding between covalently connected molecules provides a useful, potentially reversible function (e.g., stimulus-responsive binary switching) without dissociative or diffusional loss or dilution of participating binding partners. The same tethering principle is applied in hybridization-based multimolecular switches comprising two (or more) pairs of complementary defined sequence segments, all four constituent defined sequence segments being covalently attached to one another within a single discrete structure, wherein either one pair or the other is hybridized at any given time. Such tethered specific recognition devices may be nucleotide-based (i.e., relying on nucleotides for molecular positioning), or they may be constructed using a nonnucleotide scaffold, preferably a copolymer or heteropolymer or flexible polymer comprising folds, bends, joints, hinges or branchpoints. Nucleotide-directed functional coupling between selected molecules or specific recognition pairs can be used as a screening and selection criterion for identification of defined sequence segments with desired recognition properties.